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      Correlated Occurrence and Bypass of Frame-Shifting Insertion-Deletions (InDels) to Give Functional Proteins

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          Short insertions and deletions (InDels) comprise an important part of the natural mutational repertoire. InDels are, however, highly deleterious, primarily because two-thirds result in frame-shifts. Bypass through slippage over homonucleotide repeats by transcriptional and/or translational infidelity is known to occur sporadically. However, the overall frequency of bypass and its relation to sequence composition remain unclear. Intriguingly, the occurrence of InDels and the bypass of frame-shifts are mechanistically related - occurring through slippage over repeats by DNA or RNA polymerases, or by the ribosome, respectively. Here, we show that the frequency of frame-shifting InDels, and the frequency by which they are bypassed to give full-length, functional proteins, are indeed highly correlated. Using a laboratory genetic drift, we have exhaustively mapped all InDels that occurred within a single gene. We thus compared the naive InDel repertoire that results from DNA polymerase slippage to the frame-shifting InDels tolerated following selection to maintain protein function. We found that InDels repeatedly occurred, and were bypassed, within homonucleotide repeats of 3–8 bases. The longer the repeat, the higher was the frequency of InDels formation, and the more frequent was their bypass. Besides an expected 8A repeat, other types of repeats, including short ones, and G and C repeats, were bypassed. Although obtained in vitro, our results indicate a direct link between the genetic occurrence of InDels and their phenotypic rescue, thus suggesting a potential role for frame-shifting InDels as bridging evolutionary intermediates.

          Author Summary

          Homonucleotide repeats are exceptionally prone to insertions and/or deletions of bases (InDels). However, unless they occur in a multiplicity of 3 bases, InDels disrupt the reading frame and are thus expected to be purged from coding regions. Homonucleotide repeats, however, are also vulnerable to slippage by RNA polymerases and the ribosome. Using laboratory evolution techniques, we systematically mapped the occurrence of InDels within a given gene, before and after selection. Our data indicate that frame-shifting InDels were frequently bypassed to give functional proteins at surprisingly high frequencies. Further, we found a strict correlation between the repeat length, the frequency of occurrence of InDels at the DNA level, and the likelihood of bypass by transcriptional/translational slippage. Our results suggest that frame-shifting InDels might comprise functional evolutionary intermediates, and an effective mean of sequence divergence ( e.g. when an adjacent InDel restores the frame, resulting in altered sequence and, potentially, in an altered protein structure).

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          Most cited references 42

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          A genome-wide view of the spectrum of spontaneous mutations in yeast.

          The mutation process ultimately defines the genetic features of all populations and, hence, has a bearing on a wide range of issues involving evolutionary genetics, inheritance, and genetic disorders, including the predisposition to cancer. Nevertheless, formidable technical barriers have constrained our understanding of the rate at which mutations arise and the molecular spectrum of their effects. Here, we report on the use of complete-genome sequencing in the characterization of spontaneously arising mutations in the yeast Saccharomyces cerevisiae. Our results confirm some findings previously obtained by indirect methods but also yield numerous unexpected findings, in particular a very high rate of point mutation and skewed distribution of base-substitution types in the mitochondrion, a very high rate of segmental duplication and deletion in the nuclear genome, and substantial deviations in the mutational profile among various model organisms.
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            An initial map of insertion and deletion (INDEL) variation in the human genome.

            Although many studies have been conducted to identify single nucleotide polymorphisms (SNPs) in humans, few studies have been conducted to identify alternative forms of natural genetic variation, such as insertion and deletion (INDEL) polymorphisms. In this report, we describe an initial map of human INDEL variation that contains 415,436 unique INDEL polymorphisms. These INDELs were identified with a computational approach using DNA re-sequencing traces that originally were generated for SNP discovery projects. They range from 1 bp to 9989 bp in length and are split almost equally between insertions and deletions, relative to the chimpanzee genome sequence. Five major classes of INDELs were identified, including (1) insertions and deletions of single-base pairs, (2) monomeric base pair expansions, (3) multi-base pair expansions of 2-15 bp repeat units, (4) transposon insertions, and (5) INDELs containing random DNA sequences. Our INDELs are distributed throughout the human genome with an average density of one INDEL per 7.2 kb of DNA. Variation hotspots were identified with up to 48-fold regional increases in INDEL and/or SNP variation compared with the chromosomal averages for the same chromosomes. Over 148,000 INDELs (35.7%) were identified within known genes, and 5542 of these INDELs were located in the promoters and exons of genes, where gene function would be expected to be influenced the greatest. All INDELs in this study have been deposited into dbSNP and have been integrated into maps of human genetic variation that are available to the research community.
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              Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution.

              Restriction-modification (RM) systems are composed of genes that encode a restriction enzyme and a modification methylase. RM systems sometimes behave as discrete units of life, like viruses and transposons. RM complexes attack invading DNA that has not been properly modified and thus may serve as a tool of defense for bacterial cells. However, any threat to their maintenance, such as a challenge by a competing genetic element (an incompatible plasmid or an allelic homologous stretch of DNA, for example) can lead to cell death through restriction breakage in the genome. This post-segregational or post-disturbance cell killing may provide the RM complexes (and any DNA linked with them) with a competitive advantage. There is evidence that they have undergone extensive horizontal transfer between genomes, as inferred from their sequence homology, codon usage bias and GC content difference. They are often linked with mobile genetic elements such as plasmids, viruses, transposons and integrons. The comparison of closely related bacterial genomes also suggests that, at times, RM genes themselves behave as mobile elements and cause genome rearrangements. Indeed some bacterial genomes that survived post-disturbance attack by an RM gene complex in the laboratory have experienced genome rearrangements. The avoidance of some restriction sites by bacterial genomes may result from selection by past restriction attacks. Both bacteriophages and bacteria also appear to use homologous recombination to cope with the selfish behavior of RM systems. RM systems compete with each other in several ways. One is competition for recognition sequences in post-segregational killing. Another is super-infection exclusion, that is, the killing of the cell carrying an RM system when it is infected with another RM system of the same regulatory specificity but of a different sequence specificity. The capacity of RM systems to act as selfish, mobile genetic elements may underlie the structure and function of RM enzymes.

                Author and article information

                Role: Editor
                PLoS Genet
                PLoS Genet
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                October 2013
                October 2013
                24 October 2013
                : 9
                : 10
                [1 ]Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel
                [2 ]Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
                Cell and Molecular Biology, Sweden
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: LRS DST. Performed the experiments: LRS AS. Analyzed the data: LRS ÁTP OW RS. Contributed reagents/materials/analysis tools: LRS ÁTP OW RS. Wrote the paper: LRS DST.


                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 12
                Financial support by the Israel Science Foundation, and by the Meil de Botton Aynsley Foundation, are gratefully acknowledged. DST is the Nella and Leon Benoziyo Professor of Biochemistry. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article



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